We use mass spectrometry to simultaneously measure the identities and concentrations of the ~300 most abundant hair-bundle proteins, which together make up the specialized mechanically sensitive structure of sensory hair cells. Taking advantage of our ability to purify hair bundles of the chicken vestibular system with high yield and excellent purity, we will apply techniques used in systems biology, including proteomic and microarray analysis of many molecules in parallel, to understand the mechanisms of bundle development and transduction-apparatus assembly. In Aim 1, we will further characterize the concentrations and phosphorylation of already-identified bundle proteins using new mass-spectrometry experiments. As a test case, we will elevate cAMP levels in bundles to examine protein redistribution and phosphorylation. In this aim, we will also systematically study the locations of novel proteins identified in bundles. In Aim 2, we will monitor the expression of mRNAs and proteins that participate in formation of the hair bundle, analyzing chicken basilar papilla during the stages of development identified by Lew Tilney. By determining when molecules involved in bundle assembly are expressed or regulated, we will gain better mechanistic understanding of construction of the bundle. Finally, in Aim 3, we will examine which proteins increase or decrease in abundance in hair bundles following tip-link ablation with calcium chelators. These experiments will allow us to probe the mechanism of assembly of the transduction apparatus. Carrying out these three aims will allow us to take a systems-level view of the hair bundle. Moreover, application of systems-biology methodology to the bundle has a significant advantage over application to whole cells: the hair bundle is significantly less complex than a whole cell or tissue, and thus the number of molecules to be analyzed is relatively small and the mechanism and models may be significantly less complicated than those of other systems. While this assertion of simplicity may not be entirely accurate, we will be able to see the coordinated response of hundreds of proteins during critical phases of bundle and transduction-apparatus assembly. By moving towards relatively unbiased experimental approaches, we gain novel insights into structures critical for hearing and balance. PUBLIC HEALTH RELEVANCE: We propose here to study what the business end of the inner ear, the hair bundle, is made out of and how it is constructed during development of an organism. Moreover, we aim to determine what proteins make up the specialized molecular machine that actually detects sound. Success in these aims will allow us to identify additional genes that, when disrupted, lead to hearing loss. More significantly, these experiments will allow us to design rational approaches to detecting and ameliorating hearing loss and disrupted balance.